Process for the site-directed integration of DNA into the genome of plants

Abstract

The present invention provides a method for site-directed integration of DNA-sequences into the genome of plants via homologous recombination, by transforming said plants using the DNA-transfer system of Agrobacterium, in which the transforming DNA comprises in its most simple form a region homologous to the target locus, as well as a region which is different from the target locus either next to one or between two T-DNA borders. Special constructs are provided, which in its most complete form have the following general structure, <IMAGE> in which box 1 and 7 represent T-DNA borders, boxes 2 and 6 comprise functional expression cassettes containing negative selection genes, box 3 provides a region of homology with the target locus promoting recombination, box 4 represents a DNA sequence containing a mutation with respect to the target locus, box 5 represents a functional expression cassette containing a positive selection gene, and box E comprises a DNA sequence which is homologous to a region adjacent of the target locus, or in the vicinity of the target locus, which promotes homologous recombination.

We claim: 
     
       1. A process for introducing a defined DNA sequence in a selected target locus of a nuclear plant cell genome by integrating at least a part of a recombinant DNA into said genome through homologous recombination at the target locus, comprising the steps of: a) coincubating plant protoplasts or plant cells under transforming conditions, with a strain of the genus Agrobacterium capable of T-region transfer and containing a plant transformation vector which comprises recombinant DNA of the formula: ##STR6## wherein boxes 1 and 7 are T-DNA border sequences, and wherein one of box 1 or 7 may be absent; and wherein box 3 comprises a DNA sequence sufficiently homologous to a DNA sequence inside the target locus and sufficiently long to promote homologous recombination; and   wherein box 4 comprises a DNA sequence not homologous to sequences occurring in the target locus; and   wherein the lines connecting the boxes shown may comprise any number of nucleotides or basepairs, and     b) identifying protoplasts or cells that have obtained the said defined DNA sequence in their genomes at said target locus.   
     
     
       2. The process of claim 1 wherein the recombinant DNA has the formula: ##STR7## wherein box 5 comprises an expression cassette for a positive selection gene functional in said cell; and wherein box E comprises a DNA sequence sufficiently homologous to DNA sequences inside or adjacent to the target locus to promote homologous recombination.   
     
     
       3. The process according to claim 2, wherein the positive selection gene is selected from the group consisting of the nptII gene, the hpt-gene and the Als gene. 
     
     
       4. A process for introducing a defined mutation in a selected target locus of a nuclear plant cell genome by integrating at least a part of a recombinant DNA into said genome through homologous recombination at the target locus, comprising the steps of: a) coincubating plant protoplasts or plant cells under transforming conditions, with a strain of the genus Agrobacterium capable of T-region transfer and containing a plant transformation vector which comprises recombinant DNA of the formula: ##STR8## wherein boxes 1 and 7 are T-DNA border sequences, and wherein one of box 1 or 7 may be absent; and wherein box 3 comprises a DNA sequence sufficiently homologous to a DNA sequence inside the target locus and sufficiently long to promote homologous recombination; and   wherein box 4 comprises a DNA sequence sufficiently homologous to promote homologous recombination with a corresponding sequence in the target locus and wherein box (4) comprises the said defined mutation; and   wherein the lines connecting the boxes shown may comprise any number of nucleotides or basepairs, and     b) identifying protoplasts or cells that have obtained the said defined mutation in their genome at the said target locus.   
     
     
       5. The process of claim 4 wherein said recombinant DNA has the formula: ##STR9## wherein each of boxes 2 and 6 comprises an expression cassette for a negative selection gene functional in said plant cell; and wherein one of box 2 and 6 may be absent.   
     
     
       6. The process of claim 5, wherein said recombinant DNA has the formula: ##STR10## wherein box E comprises a DNA sequence sufficiently homologous to DNA inside or adjacent to the target locus to promote homologous recombination. 
     
     
       7. The process of claim 5 wherein said recombinant DNA has the formula: ##STR11## wherein box 5 comprises an expression cassette for a positive selection gene which is functional in said plant cell. 
     
     
       8. The process according to claim 7, wherein the positive selection gene is selected from the group consisting of the nptII gene, the hpt-gene and the Als-gene. 
     
     
       9. A recombinant DNA capable of introducing a defined mutation in a selected target locus of a nuclear plant cell genome through homologous recombination at the target locus, which recombinant DNA has the formula: ##STR12## wherein box i comprises the said defined mutation; and wherein boxes 1 and 7 are T-DNA border sequences, and wherein one of box 1 or 7 may be absent; and wherein each of boxes 3 and 4 individually is a DNA sequence sufficiently homologous to a DNA sequence inside the target locus and sufficiently long to promote homologous recombination; and   wherein the DNA sequences of the boxes 3 and 4 have the same 5'-to-3' orientation, but wherein said boxes 3 and 4 are rearranged with respect to the homologous sequences in the target locus.   
     
     
       10. The recombinant DNA of claim 9 which has the formula: ##STR13## wherein each of boxes 2 and 6 comprise an expression cassette for a negative selection gene functional in said plant cell; and wherein one of box 2 and 6 may be absent.   
     
     
       11. The recombinant DNA of claim 10 wherein said negative selection gene is selected from the group consisting of the aux-2 gene, the cytochrome p450 gene, the Adh-gene, and the tk-gene. 
     
     
       12. A replicon suitable for cloning in bacteria containing the recombinant DNA of claim 10. 
     
     
       13. A bacterium containing the replicon of claim 12. 
     
     
       14. A plant transformation vector which comprises the recombinant DNA of claim 10. 
     
     
       15. A plant transformation vector which comprises the recombinant DNA of claim 11. 
     
     
       16. A bacterial strain of the genus Agrobacterium capable of DNA transfer to plant cells which contains the plant transformation vector of claim 14. 
     
     
       17. A bacterial strain of the genus Agrobacterium capable of DNA transfer to plant cells which contains the plant transformation vector of claim 15. 
     
     
       18. A process for the preparation of a plant cell containing a defined mutation at a selected target locus of the nuclear genome through homologous recombination with a recombinant DNA containing said mutation, comprising the steps of, a) coincubating plant protoplasts or plant cells under transforming conditions, with a strain of the genus Agrobacterium according to claim 16, and   b) identifying a protoplast or cell that has obtained the said defined mutation.   
     
     
       19. A process for the preparation of a plant cell containing a defined mutation at a selected target locus of the genome, via homologous recombination, comprising the steps of: (a) coincubating plant protoplasts or plant cells under transforming conditions, with a strain of the genus Agrobacterium according to claim 17,   (b) selecting or enriching for cells which do not express any of the negative marker genes, under conditions allowing for selection, and   c) identifying a protoplast or cell that has obtained the defined mutation in its genome at the target locus.